CN105246496A - Salmonella-based vectors for cancer immunotherapy targeting Wilms' tumor gene WT-1 - Google Patents

Salmonella-based vectors for cancer immunotherapy targeting Wilms' tumor gene WT-1 Download PDF

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CN105246496A
CN105246496A CN201480023775.9A CN201480023775A CN105246496A CN 105246496 A CN105246496 A CN 105246496A CN 201480023775 A CN201480023775 A CN 201480023775A CN 105246496 A CN105246496 A CN 105246496A
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cancer
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海因茨·尤贝瑙诺
马尔科·施普林格
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Si Meng Limited-Liability Co Of Wanke
Vaximm AG
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Abstract

The present invention relates to an attenuated mutant strain of Salmonella comprising a recombinant DNA molecule encoding Wilms' tumor Protein 1. In particular, the present invention relates to the use of said attenuated mutant strain of Salmonella in cancer immunotherapy.

Description

For the carrier based on salmonella of the targeting nephroblastoma gene WT-1 of cancer immunotherapy
Technical field
The present invention relates to the Attenuated Salmonella mutant of the recombinant DNA molecules containing coding nephroblastoma (Wilms ' tumor) albumen 1.Particularly, the present invention relates to the purposes of described Attenuated Salmonella mutant in immunotherapy of tumors.
Background technology
Nephroblastoma gene 1 (WT1) encoding zinc finger protein transcription factor, participates in cell proliferation and differentiation.It comprises high expressed in the neoplastic hematologic disorder of some types and multiple solid tumor in many malignant tumor.On the contrary, in the normal structure of adult, WT1 expresses the CD34 be limited in gonad, uterus, kidney, mesothelium and polytype tissue +cFU-GM.WT1 was considered to tumor suppressor gene originally.But recent evidence shows the oncogenic function of this transcription factor; The differentiation of Wt-1 negative regulation CFU-GM and promote the propagation of CFU-GM.In addition, process LAN WT1 produces immunogenicity; WT1 specific T-cells and the anti-WT1 antibody of IgG is detected in cancer patient.Therefore, WT-1 is the one candidate likely of exploitation cancer vaccine.
The human clinical trial based on the WT1 vaccine of HLA (human leucocyte antigen (HLA))-restrictive WT1 fragments of peptides is used to have been reported.Osadaetal., ClinCancerRes2009; 15:2789-2796, discloses the adenovirus vaccine of coding WT1.
Known to inventor, still there is no the report of the antibacterial tumor vaccine alive of targeting WT1.In addition, the report of the oral tumor vaccine of targeting WT1 is not still had.
The attenuated derivative of Salmonella enteritidis can be used for sending the attractive carrier of heteroantigen to immune system, because Salmonella enteritidis can be sent by by mucosal immune and per os or nose, this provides simple and safe facility compared with parenteral.In addition, salmonella bacterial strain can excite strong humoral and cellular immune response response in the level in whole body and mucosa chamber.The advantage of lower cost of batch preparation, and bacterial vaccine preparation of living is highly stable.Comprise virulent gene, regulator gene and metabolic gene by disappearance several genes and can realize attenuation.
Suddenling change that several Salmonella typhimurtums of attenuation have demonstrated in animal model by aro is the safe and efficient delivery vector of heteroantigen.
By active attenuated salmonella typhimurium strain bacterial strain by coding for antigens particularly the DNA construct of VFGE receptor protein send the method entering mice target cell and see described in WO03/073995.The people such as Niethammer (NatureMedicine2002,8 (12), 1369) prove that the attenuated salmonella typhimurium strain aroA bacterial strain SL7207 containing coding Mus VEGF R2 (VEGFR-2 or FLK-1, it is that Tumor angiogenesis is necessary) expression vector has tumor vaccine function.
But only have a kind of attenuation Salmonella enteritidis serological type strain, be called Salmonella typhi Ty21a (being abbreviated as: S.typhiTy21a), to be accepted for human body and with trade name (BernaBiotechLtd., aCrucellCompany, Switzerland; Selling license PL15747/0001 disclosed in December in 1996 16 days) sell.
The oral vaccine alive of the described better tolerance for typhoid fever is obtained through chemomorphosis by wild type lethal bacteria separated strain S.typhiTy2, and comprises the sudden change of galE gene lacks functionality and other unascertained sudden changes.It is shown as through field trial effectively and licensed in many countries after safety be antityphoid vaccine.
WT1 is the tumor antigen likely that can be used for developing cancer vaccine.The main restriction of acquisition WT1 peptide vaccine is in the past HLA-restriction and parenteral.Be starved of the cancer treatment method of improvement based on targeting WT1, not yet realize so far.
Summary of the invention
Consider prior art, a target of the present invention is to provide the cancer vaccine of new oral targeting WT1.The main advantage of the cancer vaccine of described targeting WT1 is the therapeutic choice can optimizing cancer patient.
On the one hand, the present invention relates to the attenuation mutant of salmonella, it comprises the recombinant DNA molecules containing coding nephroblastoma albumen 1 (WT1) expression cassette of at least one copy.
Attenuated Salmonella strain of the present invention demonstrates anti-tumor activity in the mouse model body of inoculation murine leukemia cell.According to the inventors knowledge, this new Attenuated Salmonella bacterial strain is the antibacterial cancer vaccine alive of first targeting WT1.In addition, Attenuated Salmonella bacterial strain of the present invention is the oral cancer vaccine of first targeting WT1.Due to WT1 process LAN in multiple neoplastic hematologic disorder and solid tumor, Attenuated Salmonella bacterial strain of the present invention has very large potentiality as universal cancer vaccine.
First in research, vaccine of the present invention (VXM06) empirical tests effectively can extend the survival that intraperitoneal implants the mice of leukaemia.Result shows, inoculation VXM06 vaccine can cause immunne response and produce the immunological memory for process LAN WT1 tumor cell.To merit attention and surprisingly, novel vaccine VXM06 is effective when relative low dose.Attenuated Salmonella mutant strain of the present invention can monotherapy application or the salmonella attenuation mutant Combination application with the other DNA molecular containing other tumor antigens of encoding.In addition, attenuation mutant of the present invention can with chemotherapy, X-ray therapy or biology cancer therapy combined administration.Use VXM06 treatment also may be effective, if patient produces resistance (chemotherapy tolerance patient) to chemotherapy.Therefore new Attenuated Salmonella bacterial strain of the present invention can be used in cancer treatment method that is new, that greatly improve.
In particular embodiments, described salmonella attenuation mutant belongs to Salmonella enteritidis kind.In particular embodiments, described salmonella attenuation mutant is Salmonella typhi Ty21a.
In particular embodiments, expression cassette is eukaryotic expression box.
In particular embodiments, WT1 is selected from the group comprising people WT1 and have the albumen at least about 80% sequence identical degree with it.
In particular embodiments, WT1 is truncate.In particular embodiments, the zinc finger protein domain disappearance of WT1.In particular embodiments, the WT1 aminoacid sequence of truncate is as shown in SEQIDNO1.
In particular embodiments, recombinant DNA molecules comprises the eukaryotic expression box of kanamycin antibiotics resistance gene, pMB1ori and the encoding human WT1 controlled by CMV promoter or the albumen especially people WT1 of truncate with it with at least 80% sequence identical degree.In particular embodiments, recombinant DNA molecules is pVAX10.hWT1 plasmid and contains the nucleotide sequence shown in SEQIDNO2.
In particular embodiments, described salmonella attenuation mutant can be used as medicament.
In particular embodiments, described salmonella attenuation mutant can be used as vaccine.
In particular embodiments, described salmonella attenuation mutant can be used for cancer immunotherapy.
In particular embodiments, cancer immunotherapy also comprises one or more the other salmonella attenuation mutants containing the recombinant DNA molecules of the expression cassette of encoding tumor-antigens and/or mesenchyma stroma of tumors antigen used and comprise at least one copy.In particular embodiments, one or more other salmonella attenuation mutants described are the Salmonella typhi Ty21a comprising eukaryotic expression box.In particular embodiments, one or more other salmonella bacterial strains described comprise the salmonella attenuation mutant of encoding human VEGFR-2.
In particular embodiments, described salmonella attenuation mutant and one or more other salmonella attenuation mutants described are used jointly.
In particular embodiments, cancer immunotherapy with chemotherapy, X-ray therapy or biology cancer therapy.
In particular embodiments, described salmonella attenuation mutant is in chemotherapy or in the radiation therapy treatment cycle or use in cancer therapy process biology.
In particular embodiments, described salmonella attenuation mutant is in chemotherapy or before the radiation therapy treatment cycle or use before cancer therapy biology.
In particular embodiments, described salmonella attenuation mutant is in chemotherapy or after the radiation therapy treatment cycle or use after cancer therapy biology.
In further embodiment, described salmonella attenuation mutant is before chemotherapy, radiation therapy treatment cycle or biology cancer therapy at least one or use in process.If implement in chemotherapy, X-ray therapy or biology cancer therapy more than one, then described salmonella attenuation mutant can before at least one of described therapy or in process or before and process in use, use in the process of at least 2 kinds of described therapy especially.
In particular embodiments, described salmonella attenuation mutant direct oral cavity is used.
In particular embodiments, described cancer selected from leukaemia, be selected from acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL) particularly, and solid tumor, be selected from pulmonary carcinoma, breast carcinoma, the esophageal carcinoma, colon cancer, colorectal carcinoma, gastric cancer, bile duct duct carcinoma, cancer of pancreas, glioblastoma multiforme, incidence cancer, synovial sarcoma, angiosarcoma, osteosarcoma, thyroid carcinoma, cervical cancer, carcinoma of endometrium, ovarian cancer, neuroblastoma, rhabdomyosarcoma, carcinoma of prostate particularly.
In particular embodiments, single dose is from about 10 5to about 10 11, particularly from about 10 6to about 10 10, more specifically from about 10 6to about 10 9, more specifically from about 10 6to about 10 8, the most particularly from about 10 6to about 10 7colony-forming units (CFU).
In particular embodiments, described salmonella attenuation mutant is used for individuation cancer immunotherapy, it comprises the tumor antigen expression pattern of assess patient and/or the step of interstitial antigen presentation pattern.
Detailed description of the invention
By reference to following to specific descriptions of the present invention and wherein comprised embodiment, more easily the present invention can be understood.
In one aspect, the present invention relates to the salmonella attenuation mutant of the recombinant DNA molecules containing coding nephroblastoma albumen (WT1) expression cassette comprising at least one copy.
In the present invention, described salmonella attenuation mutant can be used as the bacteria carrier of the recombinant DNA molecules containing coding nephroblastoma albumen (WT1) expression cassette, for being delivered in target cell by described recombinant DNA molecules.
In the present invention, term " attenuation " refer to not containing Attenuating mutations parent bacterial strain compared with subtract hypotoxic bacterial isolates.The bacterial isolates of attenuation is preferably lost its toxicity but is retained the ability of its inducing protective immunity.Attenuation comprises virulent gene, regulator gene and metabolic gene by disappearance several genes and realizes.The antibacterial of attenuation can be present in nature or can manually produce in the lab, such as, by adapting to new culture medium or cell culture or producing by recombinant DNA technology.
In the present invention, term " mutant " refers to the bacterial isolates containing sudden change in its genome.Herein, term " sudden change " refers to that nucleotide sequence changes, and comprises point mutation, insertion, disappearance, swivel base and inversion.
In the present invention, term " comprises (comprises) " or " comprising (comprising) " means " including but not limited to ".Described term is intended that open, for illustration of the existence of any described feature, element, integer, step or component, but does not get rid of one kind of multiple other features, element, integer, step, component or its existence of combining or adds.Therefore, term " comprise " comprise have more determinate term " by ... composition " and " primarily of ... form ".In one embodiment, term " comprise " as the application in the whole text use consistent, especially in the claims can by " by ... composition " replace.
In the present invention, term " recombinant DNA molecules " refers to the DNA construct of through engineering approaches, preferably comprises the DNA fragmentation of separate sources.Recombinant DNA molecules can be linear nucleic acid, or imports preferably by by the exploitation reading frame of coding WT1 the ring-type recombinant dna plasmid produced in expression vector plasmid.
In the present invention, term " expression cassette " refer to by can the adjustment sequence of control WT1 gene expression control, the nucleic acid unit that at least comprises WT1 gene.The expression cassette be included in described salmonella attenuation mutant preferably can mediate transcribing of the opening code-reading frame of the coding WT1 comprised in target cell.Expression cassette typically comprises promoter, at least 1 opening code-reading frame and transcription stop signals.
Zinc-finger protein transcription factor nephroblastoma albumen 1 is by WT1 gene code.Its C-end comprises 4 zinc-finger motifs, and N-end comprises proline rich/glutamine DNA binding structural domain.The multiple transcriptional variants produced by the variable sheer of the exon of two codings is by clear qualification.WT1 plays an important role in genitourinary system is grown, and participates in cell proliferation and differentiation.WT1 gene is the Gene segregation as responsible child's tumor of kidney nephroblastoma.It comprises high expressed in the neoplastic hematologic disorder of a few types and multiple solid tumor at Several Kinds of Malignancy.On the contrary, in adult body, the normal tissue expression of WT1 is limited to the CFU-GM in gonad, uterus, kidney, mesothelium and polytype tissue.Due to its express spectra, its tumorigenesis ability and its immunogenic potential, tumor antigen WT1 is the candidate likely of exploitation cancer vaccine.
In particular embodiments, described salmonella attenuation mutant is Salmonella enteritidis kind.In particular embodiments, described salmonella attenuation mutant is Salmonella typhi Ty21a.The Salmonella typhi Ty21a bacterial strain of attenuation is Typhoral also referred to as the active component of (by BernaBiotechLtd., aCrucellCompany, Switzerland, manufactures).It is the oral vaccine alive for typhoid fever of unique license at present.This vaccine is extensively tested, and has proved patient toxicities and to propagate in the third party be safe (Wahdanetal., J.InfectiousDiseases1982,145:292-295).Described vaccine is licensed more than 40 countries.Typhoral selling license number be PL15747/0001, sign in December in 1996 16 days.The vaccine of 1 dosage contains at least 2 × 10 9active Salmonella typhi Ty21a colony-forming units and at least 5 × 10 9non-activity Salmonella typhi Ty21a cell.
A biochemical property of Salmonella typhi Ty21a bacterial isolates is that it can not metabolize galactose.Sulfate reduction can not be sulfide by described attenuated bacteria bacterial strain, and this makes itself and wild type Salmonella typhi Ty2 bacterial strain distinguish.As for its serological property, Salmonella typhi Ty21a bacterial strain contains O9-antigen, and it is a kind of bacterial outer membrane polysaccharide; And lack O5-antigen, it is the characteristic component of Salmonella typhimurtum.Described Serological Characterization support comprises the principle of each test for discharging in batches in group's characterization test.
In particular embodiments, expression cassette is eukaryotic expression box.In the present invention, term " eukaryotic expression box " refers to can at the expression cassette of eukaryotic expression opening code-reading frame.Show, induce the amount of the heteroantigen required for enough immunne response may be toxicity for antibacterial and the over-expression decay of cell death, heteroantigen can be caused or lack.Be used in bacteria carrier and do not express but the eukaryotic expression box of only expressing in target cell can overcome described toxicity problem, and expressed albumen can demonstrate eucaryon glycosylation pattern.
Eukaryotic expression box comprises adjustment sequence, preferred promoter and the polyadenylation signal that can control opening code-reading frame and express in eukaryotic cell.Promoter in the recombinant DNA molecules that Attenuated Salmonella mutant of the present invention comprises and polyadenylation signal are preferably having activity in by the subject cell of immunity.Especially for the example of the promoter producing people's DNA vaccination, the promoter be applicable to includes but not limited to that the promoter such as strong cmv immediate early promoter from cytomegalovirus (CMV) is sub, from simian virus 40 (SV40), mouse mammary tumor virus (MMTV), promoter such as HIV long terminal repeat (LTR) promoter of human immunodeficiency virus (HIV), from moloney virus, the promoter of Epstein-Barr virus (EBV) and rous sarcoma virus (RSV), and from people's gene such as human actin, human myoglobulin, human hemoglobin, the promoter of people's flesh creatine and human metal thioalbumen gene.In a specific embodiment, eukaryotic expression box contains CMV promoter.In the present invention, term " CMV promoter " refers to strong at once early stage cytomegalovirus promoter.
The polyadenylation signal be applicable to, the example especially for the polyadenylation signal producing people's DNA vaccination includes but not limited to bovine growth hormone (BGH) polyadenylation site, SV40 polyadenylation signal and LTR polyadenylation signal.In particular embodiments, the eukaryotic expression box comprised in the recombinant DNA molecules that salmonella attenuation mutant of the present invention contains contains BGH polyadenylation site.
Except the regulating element needed for allos WT1 gene expression such as promoter and polyadenylation signal, in described recombinant DNA molecules, also can comprise other elements.This other element comprises enhancer.Enhancer can be that the enhancer of such as human actin, human myoglobulin, human hemoglobin, people's flesh creatine and virus enhancer are such as from the enhancer of CMV, RSV and EBV.
Regulate sequence and codon normally species-independent, therefore produce for maximizing albumen, adjustment sequence used and codon are preferably effective in immune species.Those skilled in the art can do the recombinant DNA molecules producing and have function in given species.
In particular embodiments, WT1 is selected from the group comprising people WT1 and have with it at least about 80% sequence identical degree albumen.
Herein, term " about " or " approximately " mean, between 80% to 120% of set-point or scope, selectively between 90% to 110%, to be included between 95% to 105%.
Herein, term " has the albumen at least about 80% sequence identical degree from people WT1 " and refers to and the aminoacid sequence of people WT1 and/or the different albumen of the nucleotide sequence of encode such amino acid sequences.Described albumen can be the homologue of the such as different plant species WT1 of nature origin, or engineered protein such as through engineering approaches WT1 derivant.Known, codon used between different plant species is different.Therefore, when during expressing heterologous albumen, adjusting nucleotide sequence and select may be necessary with the codon adapting to target cell, or be at least useful in target cell.The method designing and build known protein derivatives well known to a person skilled in the art.
Can be comprised one or more with the people WT1 albumen had at least about 80% sequence identical degree to suddenly change, comprise one or more amino acid whose insertion, disappearance and/or displacement.According to instruction of the present invention, the aminoacid of described disappearance, insertion and/or displacement can be continuous print aminoacid or can be scatter in having at least about the albumen of 80% sequence identical degree a full length amino acid sequence with people WT1.According to instruction of the present invention, can insert, lack and/or replace the aminoacid of any amount, as long as the identical degree of described sequence and people WT1 is at least about 80%.In particular embodiments, be at least about 80%, at least about 85%, at least about 90% or the most particularly at least about 95% with the sequence identical degree of people WT1.Method and algorithm for determining sequence identical degree comprise by Parent Protease with relative to parental array, there is disappearance, insert and/or its derivant of displacement to compare be well known to a person skilled in the art.At DNA level, encoding the nucleotide sequence had at least about 80% sequence identical degree albumen from people WT1 can be different to a greater degree, due to the degeneracy of genetic code.
In particular embodiments, WT1 is truncate.In particular embodiments, the Zinc finger domain of WT1 is lacked.In particular embodiments, the WT1 aminoacid sequence of truncate is as shown in SEQIDNO1.
The Zinc finger domain of WT1C-end comprises 4 zinc-finger motifs.The WT1 aminoacid sequence of the truncate shown in SEQIDNO1 represents 1 to 371 amino acids of UniProtrefP19544-7.The disappearance of Zinc finger domain minimizes the risk with other containing the immune cross-reactivity of zinc finger transcription factor.In addition, the WT1 lacking the truncate of Zinc finger domain has larger immunogenic potential than total length WT1.In addition, DNA is eliminated to the oncogenic potential of WT1 in conjunction with the disappearance of important zinc-finger motif, thus minimize carcinogenic risk.
In particular embodiments, recombinant DNA molecules comprises kanamycin antibiotics resistance gene, pMB1ori and the encoding human WT1 controlled by CMV promoter or the eukaryotic expression box with it with at least 80% sequence identical degree albumen especially people WT1 of truncate.In particular embodiments, recombinant DNA molecules is pVAX10.hWT1 plasmid, and described nucleotide sequence is as shown in SEQIDNO2.
In particular embodiments, recombinant DNA molecules is from commercially available pVAX1 tMexpression plasmid (Invitrogen, SanDiego, California).The low copy pMB1 replication origin that described expression vector is substituted by pBR322 by the pUC replication origin copied by height is modified.Carrying out described low copy modification is to lower metabolic burden and making construct more stable.The expression vector skeleton called after pVAX10 produced.By NheI/XhoI, the people WT1 of truncate is inserted described expression vector skeleton and produce the expression plasmid pVAX10.hWT1 with nucleotide sequence shown in SEQIDNO2.The plasmid figure of expression plasmid pVAX10.hWT1 as shown in Figure 3.
In particular embodiments, described salmonella attenuation mutant can be used as medicament.
In particular embodiments, described salmonella attenuation mutant can be used as vaccine.
In the present invention, after term " vaccine " refers to and uses can in experimenter the reagent of induce immune response.Vaccine can preferably prevent, alleviate or disease therapy.Vaccine of the present invention comprises salmonella attenuation mutant, preferred Salmonella typhi Ty21a.Vaccine of the present invention also comprises the recombinant DNA molecules of the preferred eukaryotic expression box of expression cassette containing coding WT1 of at least one copy, and described WT1 is preferably selected from people WT1 or has the albumen at least about 80% sequence identical degree with it.Preferably, described WT1 is truncate, preferably lacks Zinc finger domain.
The Attenuated Salmonella mutant comprising the work of the recombinant DNA molecules of coding WT1 of the present invention can be used as the carrier of recombinant DNA molecules described in oral delivery.This delivery vector comprising the DNA molecular of encoding foreign antigen such as WT1 is called DNA vaccination.
The comparable traditional vaccination of heredoimmunity is more favourable.Target DNA can detect within a very long time, therefore as the storage vault of antigen.Sequence motifs in some plasmids such as GpC island is immunostimulating, and can be used as the immunostimulating adjuvant of LPS and the generation of other cell components.
Compared with can only mediating the immunity for small fragment WT1 albumen with peptide vaccine, genetic vaccination can produce the immunity of the multiple epi-position of the WT1 full length protein existence for coding.
In addition, the WT1 peptide vaccine mostly applied in clinical trial has limited application, and this is because the HLA of described peptide limits, i.e. the binding ability of the HLA molecule of they and antigen-presenting cell (APC).On the contrary, DNA vaccination of the present invention is not by HLA restriction.
The bacteria carrier of living can produce they self immunoregulatory factor such as lipopolysaccharide (LPS) in position, and this can form the advantage using such as microencapsulation than other forms.In addition, entered prove favourable by natural route, because the M cell of many antibacterials such as salmonella by Peyer patches flows out from enteric cavity, and last migration enters lymph node and spleen, therefore can bring out site by immune for vaccine targeting.The vaccine strains Ty21a of Salmonella typhi has been proved to be at present has good safety.After flowing out enteric cavity by M cell, antibacterial is absorbed by phagocyte such as macrophage and dendritic cell.These cells are activated by pathogen and start differentiation, and may move and enter lymph node and spleen.Due to its Attenuating mutations, Salmonella typhi Ty21 strain can not retain in described phagocyte, but dead at this time point.Described recombinant DNA molecules is released, and subsequently by specificity transportation system or be transferred by endosome seepage enter phagocytic immunocyte cytosol in.Finally, described recombinant DNA molecules enters in core, and it is transcribed in core, causes the WT1 in phagocyte cytosol to express.Specific cytotoxic T cell for WT1 is induced by the antigen-presenting cell activated.
There is no data at present and show that Salmonella typhi Ty21a can enter blood system.Therefore, the attenuated salmonella Ty21a vaccine strain of described work can selectively targeted immune system, demonstrates good safety simultaneously.
The attenuated derivative of Salmonella enteritidis can be used for sending the attractive carrier of heteroantigen to immune system, because Salmonella enteritidis bacterial strain can be sent by by mucosal immune and per os or nose, this provides simple and safe facility compared with parenteral.In addition, salmonella bacterial strain can excite strong humoral and cellular immune response response at whole body and mucosa chamber level.
In particular embodiments, the attenuation mutant of described salmonella is used to cancer immunotherapy.
In particular embodiments, cancer immunotherapy also comprises one or more the other salmonella attenuation mutants containing the recombinant DNA molecules of the expression cassette of encoding tumor-antigens and/or mesenchyma stroma of tumors antigen used and comprise at least one copy.In particular embodiments, one or more other salmonella attenuation mutants described are the Salmonella typhi Ty21a comprising eukaryotic expression box.In particular embodiments, one or more other salmonella bacterial strains described comprise the salmonella attenuation mutant of encoding human VEGFR-2.
Salmonella attenuation mutant of the present invention is combined with other attenuation mutants of the DNA molecular containing other tumor antigen of encoding and can has synergistic antitumor effect.Particularly, the tumor antigen that targeting is different simultaneously can minimize the risk of tumor escape.Cancer immunotherapy based on WT1 is combined with the immunization therapy based on VEGFR-2 and can produces special effect, because the tumor cell of WT1 process LAN and tumor vascular system can be attacked simultaneously.
In particular embodiments, described salmonella attenuation mutant and one or more other salmonella attenuation mutants described are used jointly.
In the present invention, term " is used (co-administration) " jointly or " jointly using (co-administer) " means more specifically more specifically in 12 hours, more specifically using two kinds of different salmonella attenuation mutants on the same day at continuous 2 days for three days on end.The most particularly, in the present invention, term " is jointly used (co-administration) " and is referred to and use two kinds of different salmonella attenuation mutants simultaneously.
In particular embodiments, cancer immunotherapy along with chemotherapy, X-ray therapy or biology cancer therapy.For curing cancer, it may be required for removing cancer stem cell completely.For maximum efficiency, it may be favourable for being combined by different Therapeutic Method.
In the present invention, term " cancer therapy biology " or " immunotherapy for cancer " refer to and excite the immune system of patient to attack malignant cell or mesenchyma stroma of tumors.Cancer therapy comprised and sent tumor antigen, delivery treatments antibody as medicine biology, used immunostimulatory cells Summing Factor and used immunocyte.
The chemotherapeutant that can combinationally use with salmonella attenuation mutant of the present invention can be such as: gemcitabine, amifostine (ethyol), Cabazitaxel, cisplatin, dacarbazine (DTIC), dactinomycin, docetaxel, chlormethine, streptozotocin, cyclophosphamide, carrnustine (BCNU), lomustine (CCNU), doxorubicin (amycin), Mycocet (doxil), folinic acid, gemcitabine (gemzar), daunorubicin, daunorubicin liposome (daunoxome), procarbazine, ketoconazole, mitomycin, cytosine arabinoside, etoposide, methotrexate, 5-fluorouracil (5-FU), vinblastine, vincristine, bleomycin, paclitaxel (taxol), docetaxel (taxotere), aldesleukin, asparaginase, busulfan, carboplatin, cladribine, camptothecine, CPT-11, 10-hydroxyl-7-Ethyl-camptothecin (SN38), dacarbazine, floxuridine, fludarabine, hydroxyurea, different cyclophosphamide, idarubicin, mesna, interferon-alpha, interferon-β, irinotecan, mitoxantrone, hycamtin, leuprorelin, megestrol, melphalan, mercaptopurine, oxaliplatin, plicamycin, mitotane, pegaspargase, pentostatin, pipobroman, plicamycin, streptozotocin, tamoxifen, teniposide, testolactone, thioguanine, phosphinothioylidynetrisaziridine, uracil mustard, vinorelbine, benzenebutanoic acid ammonia mustard and combination thereof.
In the present invention, the most preferred chemotherapeutant combined with VXM06 is Cabazitaxel, carboplatin, oxaliplatin, cisplatin, cyclophosphamide, docetaxel, gemcitabine, doxorubicin, paclitaxel (taxol), irinotecan, vincristine, vinblastine, vinorelbine, folinic acid, 5-fluorouracil and bleomycin, particularly gemcitabine.
According to contingent side effect, it also can be favourable for comprising the treatment using antibiotic or antiinflammatory.
If the side effect occurred is similar to the allergy that histamine, leukotrienes or cytokine class cause, the treatment option of, bronchospasm fixed for fever, anaphylaxis, unstable blood pressure and respiratory disorder can be used.Occur the unwanted autoaggression from T-cell treatment option can with reference to after stem cell transplantation for acute and standard regimens that is chronic graft versus host disease.Advise cyclosporin and glucocorticoids as treatment option.
In the general Ty21a type infection by Salmonella typhi situation unlikely occurred, the antibiotic therapy that recommendation is suitable, such as, use Fluoroquinolones to comprise ciprofloxacin or Ofloxacin.Gut bacteria infects and can use the such as rifaximin treatment of corresponding reagent.
In particular embodiments, described salmonella attenuation mutant is in chemotherapy or in the radiation therapy treatment cycle or use in cancer therapy process biology.
In particular embodiments, described salmonella attenuation mutant is in chemotherapy or before the radiation therapy treatment cycle or use before cancer therapy biology.The method has such advantage, implements under the condition that namely chemotherapy or X-ray therapy can strengthen in cancer immunity.
In particular embodiments, described salmonella attenuation mutant is in chemotherapy or after the radiation therapy treatment cycle or use after cancer therapy biology.
In particular embodiments, described salmonella attenuation mutant is Orally administered.Orally administered simpler than parenteral administration, safer and more comfortable.WT1 peptide vaccine mostly for clinical trial is normally subcutaneous or intradermal administration, often can cause skin erythema and local inflammatory response.Described side effect can be overcome by Orally administered DNA vaccination of the present invention.But the approach that salmonella attenuation mutant of the present invention is also applicable to by other is used.Preferably, give experimenter by treatment effective dose, and this dose-dependant is in the body weight of the type of concrete application, malignant tumor, experimenter, age, sex and health status, the mode used and dosage form etc.Using can be single or multiple, as required.
Salmonella attenuation mutant of the present invention can solution, suspension, lyophilized preparation or arbitrarily other be applicable to forms provide.It can provide with pharmaceutically acceptable carrier, diluent and/or excipient composition.Also can comprise for regulating the reagent of pH, buffer agent, for regulating the reagent etc. of toxicity.In the present invention, term " pharmaceutically acceptable " refers to the pharmaceutical formulation when giving mammal (such as people) and does not typically produce molecular substance and the other drug composition components of untoward reaction.Term " pharmaceutically acceptable " also means by administrative organization's accreditation of federal or state government or lists American Pharmacopeia in or other pharmacopeia of generally acknowledging can be used for mammal and more specifically for people's.
In particular embodiments, described cancer is selected from acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL), and solid tumor is selected from pulmonary carcinoma, breast carcinoma, the esophageal carcinoma, colon cancer, colorectal carcinoma, gastric cancer, bile duct duct carcinoma, cancer of pancreas, glioblastoma multiforme, incidence cancer, synovial sarcoma, angiosarcoma, osteosarcoma, thyroid carcinoma, cervical cancer, carcinoma of endometrium, ovarian cancer, neuroblastoma, rhabdomyosarcoma, carcinoma of prostate particularly.
Vaccine of the present invention is effective surprisingly when relatively low dosage.In particular embodiments, single dose is from about 10 5to about 10 11, particularly from about 10 6to about 10 10, more specifically from about 10 6to about 10 9, more specifically from about 10 6to about 10 8, the most particularly from about 10 6to about 10 7colony-forming units (CFU).The bacterial vaccine of the present invention alive using low dosage can minimize excretion and therefore transfer to the risk of the third party.
Herein, within term " about " or " approximately " mean set-point or scope 3 times or within 2 times, comprise within 1.5 times.
In particular embodiments, salmonella attenuation mutant can be used for personalized oncotherapy, comprises the step of evaluate patient tumor antigen expression pattern and/or interstitial antigen presentation pattern.
VXM06 can be used alone or combines with other cancer vaccines containing eukaryotic expression system based on Salmonella typhi Ty21a and is used for the treatment of polytype cancer.In particular embodiments, VXM06 can be used for the special treatment of cancer of personalized patient.For this purpose, can in the tumor of first step evaluate patient and/or interstitial antigen presentation pattern, such as by with diagnosis target to the special tumor of patient and/or interstitial antigen presentation pattern.According to the tumor of patient and/or interstitial antigen presentation pattern, VMX06 can be used separately or its and one or more applicable other are contained the cancer vaccine combined administration based on Salmonella typhi Ty21a of eukaryotic expression system.In addition, VXM06 and one or more other also can be used as fixed Combination based on the combination of the cancer vaccine of Salmonella typhi Ty21a and use.Can by independent ready-made grouping of commodities containing two or more intermixtures based on the cancer vaccine of Salmonella typhi Ty21a.Described combination, no matter fixed Combination or personalized combination, can comprise VXM01 for angiogenesis inhibitor Primary Care.
Accompanying drawing explanation
The aminoacid sequence of the people WT1 of the truncate of being encoded by WT1cDNA comprised in Fig. 1: pVAX10.hWT1 plasmid;
The nucleotide sequence of Fig. 2: pVAX10.hWT1;
The plasmid map of Fig. 3: pVAX10.hWT1;
Fig. 4: suffer from the Kaplan-Meier survival curve after FBL-3 leukemic mice use VXM0m_empty, VXM06m and VXM06 treatment;
Fig. 5: suffer from the average viability after FBL-3 leukemic mice use VXM0m_empty, VXM06m and VXM06 treatment;
Table 1: outer-gene synthesizes: phosphorylation reaction scheme;
Table 2: outer-gene synthesizes: amplification connects product-PCR overview;
Table 3: vaccine combination;
Table 4: evaluate anti-tumor activity-experimental design;
Table 5: suffer from the average viability after FBL-3 leukemic mice use VXM0m_empty, VXM06m and VXM06 treatment;
Table 6: the dead animal number of process in time after tumor inoculation (tumorchallenge);
Table 7: suffer from the average viability after FBL-3 leukemic mice use VXM0m_empty, VXM06m and VXM06 treatment and meta survival rate;
embodiment
the preparation of embodiment 1 recombiant plasmid pVAX10.mWT1 and pVAX10.hWT1
By the people WT1 (1116bp of truncate, WT1 sequence is according to reference sequences P19544-7 in UniProt data base, clip Zinc finger domain) and the Mus WT1 (1101bp of truncate, WT1 sequence is according to reference sequences P22561-5 in UniProt data base, clips Zinc finger domain) clone in the pVAX10 skeleton derived by pVAX10.VR2-1.WT1DNA fragment synthesizes generation by double-stranded gene, and wherein the thermally-stabilised ligase of oligonucleotide couples together.
the design of oligonucleotide and synthesis:
The first step, uses " SeqEditor " (Entelechon) software that Mus WT1 (the clipping Zinc finger domain) gene order of the people WT1 of truncate and truncate is divided into the independent oligonucleotide of 40 to 50 bases.Described oligonucleotide is overlapping also corresponds to two DNA chains.After the oligonucleotide of synthesis two DNA chains, oligonucleotide 10mMTris (pH8.5) is diluted to the final concentration of 50pmol/ μ l.
kinase reaction:
Then the oligonucleotide of external synthesis and T4 polynucleotide kinase are hatched and make its phosphorylation, to be connected by described oligonucleotide subsequently.By the oligonucleotide phosphorylation of positive-sense strand and antisense strand.
Reaction scheme is summarized as follows table 1:
Reactant mixture is hatched 1 hour in 37 DEG C of water-baths.Then at 95 DEG C heating within 5 minutes, make T4 polynucleotide kinase inactivation, then immediately with ice-cooled until next step process.
Kinase Mix described in 12.5 μ L is directly used in connection (20UTaqDNA ligase, 35 μ l reaction volume (NewEnglandBiolabs, M0208S).
(1 DEG C/min) is cooled gradually after 95 DEG C of degeneration.In the process, complementary oligonucleotide is assembled into double-stranded DNA.Described process completes in thermal cycler (PersonalCycler, Biometra).By adding thermostable Taq DNA ligase, by 5 '-PO of described oligonucleotide 4-end is connected with the free 3 '-OH-end of subsequent oligonucleotide acid.By repeating denature and renature step, the oligonucleotide of mispairing is discharged.Program is last, by mixture 4 DEG C of coolings.
product is connected by pcr amplification
Use flank primers to carry out pcr amplification in 50 μ l systems the connection product (about 1.1kb) that 5 μ l obtain, step is as following table 2:
PCR mixture comprises following component: 0.2 – 0.5 μM often kind of primer, 20mMTris-Cl, pH8.8,10mMKCl, 10mM (NH 4) 2sO 4, 2mMMgSO 4, 0.1%TritonX-100,25mMdNTP (often kind), 2UVent rpolymerase.
The DNA fragmentation (people of truncate and Mus WT1, respectively about 1.1kb) of external synthesis is entered (the VEGFR-2 coding region of recombiant plasmid pVAX10.VR2-1 is replaced by the people of truncate or Mus WT1) in pVAX10 skeleton through NheI/XhoI clone.For quality control, after being transformed into escherichia coli by the order-checking of whole plasmid and with corresponding canonical sequence comparison.Two sequences are all proved to be not containing mistake.By plasmid called after pVAX10.mWT1 and pVAX10.hWT1 of gained.
embodiment 2: use recombinant plasmid transformed Attenuated Salmonella bacterial strain
Salmonella typhi Ty21a is transformed with plasmid pVAX10.hWT1.Salmonella typhi SL7207 (aroA is transformed with plasmid pVAX10.mWT1 -).Transformed by electroporation.
prepare salmonella competent cell:
The glycerol culture of Salmonella typhi Ty21a and Salmonella typhi SL7207 is inoculated into LB flat board (not containing the soy peptone of animal component (ACF)).Dull and stereotyped 37 DEG C of overnight incubation.Each bacterium colony is used for overnight liquid preculture.Every 3mlLB culture medium (ACF soy peptone) inoculates a bacterium colony, overnight incubation under 37 DEG C and 180rpm condition.For preparing competent cell, 2 × 300mlLB culture medium (ACF soy peptone) being inoculated by overnight culture described in 3ml and hatches until OD under 37 DEG C and 180rpm condition 600about 0.5.Then culture is placed 10 minutes on ice.Subsequently, by gained antibacterial 3000 × g, at 4 DEG C centrifugal 10 minutes, every group precipitation thing is resuspended in the ice-cold H of 500mL 2o destin.After recentrifuge, bacterial precipitation thing 10% ice-cold glycerol is washed twice.Two group precipitation things are concentrated in 2ml10% glycerol, and final freezing on dry ice with 50 μ l deciles.Glycerol used does not comprise any animal component (SigmaAldrich, G5150).
transform salmonella competent cell:
For each conversion reaction, point competent cells such as 50 μ l are thawed 10 minutes on ice.Add 3-5 μ l plasmid DNA (pVAX10.hWT1 is used for the competent cell of Salmonella typhi Ty21a, and pVAX10.mWT1 is used for the competent cell of salmonella typhi SL7207), then mixture is hatched 5 minutes on ice.Then, mixture is transferred to the cuvette (1 mm of thickness) cooled in advance.Electric pulse is carried out with 12.5kV/cm.Then immediately 1mlLB culture medium (ACF soy peptone) is joined in described cell, cell is transferred in 2mlEppendorf pipe 37 DEG C of vibrations 1 hour.In desk centrifuge after of short duration centrifugal (16600rcf, 20s), bacterial precipitation thing is resuspended in not containing in antibiotic 200 μ lLB (ACF soy peptone) culture medium.Drigalski spreader is used to be coated on by gained mixture on the LB flat board (ACF soy peptone) containing kanamycin (concentration=25 μ g/ml or 50 μ g/ml).By flat board in 37 DEG C of overnight incubation.
the plasmid preparation of restructuring salmonella clone:
3ml is cloned in containing 37 DEG C of overnight incubation in the LB culture medium (ACF soy peptone) of kanamycin (50 μ g/ml) by 3 of often kind of restructuring salmonella bacterial strain.Then by centrifugal (16600rcf, 30s) precipitum culture.The NucleoSpin plasmid kit from Macherey-Nagel is used to carry out plasmid separation.With 50 μ l water by plasmid DNA from silicagel column elution.5 μ l eluents in agarose gel with comparing.
For long term storage, prepare the 1ml glycerol culture of positive colony.In order to this object, in 1 low ml spiral microtubule, 172 μ l glycerol (not containing animal component) are joined in 828 μ l culture medium of the 3ml culture of exponential growth.By sample storage-70 DEG C for after.
the full order-checking of the recombinant plasmid dna be separated from salmonella:
3ml liquid LB-kanamycin culture medium (ACF soy peptone) is inoculated with the restructuring salmonella of a bacterium colony (carry the Salmonella typhi Ty21a of pVAX10.hWT1 and carry the Salmonella typhi SL7207 of pVAX10.mWT1), and 37 DEG C, overnight incubation under 180rpm.By overnight culture in desk centrifuge (Biofugepico, Heraeus) with the centrifugal 30s of 1300rpm.Plasmid separation is carried out with the NucleoSpin plasmid kit from Macherey-Nagel.After the genomic DNA precipitating high molecular and cellular component, plasmid DNA is loaded onto the post with pellosil through alkaline lysis.After washing, with 50 μ l sterilized water by plasmid eluting checking order from post.Then by the comparison that bacterium colony is special, will record sequence and the comparison one by one of corresponding canonical sequence, namely the plasmid sequence of each salmonella clone is compared with canonical sequence one by one.All sequences all meets corresponding canonical sequence.Gained restructuring salmonella Strain Designation is VXM06 (carrying the salmonella Ty21a of pVAX10.hWT1 plasmid) and VXM06m (carrying the salmonella SL7207 of pVAX10.mWT1 plasmid).
embodiment 3: the anti-tumor activity evaluating VXM06 and VXM06m in homology leukemia mouse model
In homology leukemia C57/BL6J mouse model through 43 days evaluate VXM06 and VXM06m usefulness (next day dosage give, totally 4 times, and the 17th day inoculation leukaemia).Two groups of male mices, often organize 10 (n=10), respectively with 10 10the dosage of CFU/ time accepts VXM06 (Salmonella typhi Ty21a, the pVAX10.hWT1 of the people WT1 containing codified truncate) or VXM06m (Salmonella typhi, the pVAX10.mWT1 of the Mus WT1 containing codified truncate).The matched group of similar formation gives VXM0m_empty (not containing the Salmonella typhi vehicle Control of expression plasmid), and dosage is identical with processed group.In this research, carry out body weight, mortality rate and survival rate and measure.Before formal experiment, use 5.0 × 10 6with 3.0 × 10 7individual FBL-3 cell carries out the preliminary experiment of 14 days in not vaccinated male C57B16 mice (often organizing n=5), then show that previous cell concentration is for preferred in formal vaccine experiments.
dNA vaccination:
By VXM06, VXM06m and not containing the Salmonella typhi empty carrier (VXM0m_empty) of expression plasmid be stored in≤-80 DEG C for subsequent use.The bottle used in inoculation is no longer freezing, but discards subsequently.Under in research, the feature of DNA vaccination used is shown in table 3:
administration routes:
Every animal uses 100 μ lVXM0m_empty, VXM06m and VXM06 at every turn.Test event is used after melting within 30min.Used test project is given by intubate direct oral cavity gavage (per os, P.O), and volume injected is 100 μ l/ mices.
Do not consider that animal divides into groups, every animal accepts predose and uses buffer agent with the acid medium in middle stomach function regulating, then administration (giving 100 μ l/ animals/use to all dosage groups).Described buffer agent is containing 2.6g sodium bicarbonate, 1.7gL-ascorbic acid, 0.2g lactose monohydrate and 100ml drinking water.Before using described test event, carry out predose and use nearly 30 minutes.
cell culture:
The mouse leukemia cell FBL-3 of process LAN WT1 needs the cell going down to posterity to ensure survival rate and obtain aequum.
Collect the tumor cell of exponential phase of growth, it is mixed (to advise dilution factor 1:1) with trypan blue and measure for survival rate, and use counting chamber manual count by optical microscope.FBL-3 cell is washed and is resuspended in and do not enter C57BL/6 mice containing being used for injection in the RPMI medium of serum.
vaccine and tumor cell inoculation:
The cell infusion condition that intraperitoneal (I.P.) is injected: cell survival rate>=97%; 5.0 × 10 6individual cell/500 μ l/ mice.
By animal (30 C57BL/6 mices, 4-6 age in week, male, ≈ 20g was every only, CharlesRiver, France) numbering, give unique animal identification ear otch labelling, twice measured body weight weekly.
Every 10 mices (n=10 is male) are inoculated with VXM0m_empty, VXM06m and VMX06.At the 1st, 3,5 and 7 day, by 10 10cFU/ uses the vaccination of direct oral cavity gavage.17th day, by I.P. path by FBL-3 tumor cell inoculation in mice.Under experimental design summary is shown in table 4:
Result:
Under survival rate data in 3 process is shown in table 5:
The all animals of clinical examination every day, comprising: behavior, illness symptom (in cachexia, progress and mobile or diet is difficult).
Under after tumor challenge, animal dead number is shown in table 6:
Fig. 4 shows the Kaplan-Meier survival curve suffering from FBL-3 leukemia mouse with VXM0m_empty, VXM06m and VXM06 process.Compared with control mice, use the mice of VXM06m and VXM06 process can survive more for a long time (reaching 26 days).The mouse survival of the mice comparable VXM0m_empty process of about 40%VXM06m and VXM06 process is more of a specified duration.Under the meansigma methods of 3 test group survival rates and intermediate value are shown in table 7:
Fig. 5 shows the average viability that VXM0m_empty, VXM06m and VXM06 process suffers from FBL-3 leukemia mouse.Compared with control mice, use the mice of VXM06m and VXM06 process can survive more of a specified duration.
The effect of WT1 process LAN leukaemia in this experiment display VXM06 and VXM06m construct targeting mouse model.With control mice maximum survival 16 days (see Fig. 4) after tumor cell inoculation of empty carrier process.But the mice of VXM06m or VXM06 process demonstrates the survival rate of prolongation compared with the mice of control vector VXM0m_empty immunity (two kinds of test events are all 26 days).The matched group survival of the mice ratio VXM0m_empty process of about 40%VXM06m or VXM06 process is more of a specified duration.
In a word, VXM06m and VXM06 demonstrates the drug effect to test animal survival rate in homology C57B16 leukemia mouse model.Compared with empty carrier, VXM06m with VXM06 two kinds of compounds demonstrate similar drug effect.Described result shows, vaccine of the present invention can start the response for WT1 in immunocompetent mouse leukemia model, causes surviving more for a long time compared with the animal of empty carrier process.

Claims (15)

1. a salmonella attenuation mutant, it comprises the recombinant DNA molecules containing coding nephroblastoma albumen 1 (WT1) expression cassette of at least one copy.
2. salmonella attenuation mutant as claimed in claim 1, wherein said salmonella attenuation mutant is Salmonella enteritidis kind, and especially, wherein said salmonella attenuation mutant is Salmonella typhi Ty21a.
3. salmonella attenuation mutant as claimed in claim 1 or 2, wherein said expression cassette is eukaryotic expression box.
4. the salmonella attenuation mutant as described in any one of Claim 1-3, the group that the albumen that wherein WT1 selects freeman WT1 and has at least 80% sequence identical degree with it forms, especially, wherein WT1 is truncate, more particularly, wherein the Zinc finger domain of WT1 is disappearance, and the most especially, the aminoacid sequence of the WT1 of wherein said truncate is as shown in SEQIDNO.1.
5. the salmonella attenuation mutant as described in claim 3 or 4, wherein said recombinant DNA molecules comprises the eukaryotic expression box of kanamycin antibiotics resistance gene, pMB1ori and the encoding human WT1 controlled by CMV promoter or the albumen especially people WT1 of truncate with it with at least 80% sequence identical degree.
6. the salmonella attenuation mutant as described in any one of claim 1 to 5, it can be used as medicament.
7. salmonella attenuation mutant as claimed in claim 6, it can be used as vaccine.
8. salmonella attenuation mutant as claimed in claim 7, it can be used for cancer immunotherapy.
9. salmonella attenuation mutant as claimed in claim 8, wherein cancer immunotherapy also comprises one or more the other salmonella attenuation mutants containing the recombinant DNA molecules of the expression cassette of encoding tumor-antigens and/or mesenchyma stroma of tumors antigen used and comprise at least one copy, especially, one or more other salmonella attenuation mutants wherein said are the Salmonella typhi Ty21a comprising eukaryotic expression box, more particularly, one or more other salmonella attenuation mutants wherein said comprise the salmonella attenuation mutant of encoding human VEGFR-2, more particularly, one or more other salmonella attenuation mutants wherein said comprise the salmonella attenuation mutant of VXM01 by name.
10. as described in aforementioned any one claim, particularly salmonella attenuation mutant according to claim 9, wherein said salmonella attenuation mutant and one or more other salmonella attenuation mutants described are used jointly.
11. salmonella attenuation mutants as described in any one of claim 8 to 10, wherein cancer immunotherapy with chemotherapy, X-ray therapy or biology cancer therapy, especially, wherein said salmonella attenuation mutant is in described chemotherapy or before the radiation therapy treatment cycle or in its process or before biology cancer therapy or in its process or before described chemotherapy or radiation therapy treatment cycle or biology cancer therapy or use in its process.
12. salmonella attenuation mutants as described in any one of claim 6 to 11, wherein said salmonella attenuation mutant is oral administration.
13. salmonella attenuation mutants as described in any one of claim 8 to 12, wherein said cancer selected from leukaemia, be selected from acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL) especially, and be selected from solid tumor, be selected from pulmonary carcinoma especially, breast carcinoma, the esophageal carcinoma, colon cancer, colorectal carcinoma, gastric cancer, bile duct duct carcinoma, cancer of pancreas, glioblastoma multiforme, incidence cancer, synovial sarcoma, angiosarcoma, osteosarcoma, thyroid carcinoma, cervical cancer, carcinoma of endometrium, ovarian cancer, neuroblastoma, rhabdomyosarcoma, carcinoma of prostate.
14. salmonella attenuation mutants as described in any one of claim 6 to 13, wherein single dose is from about 10 5to about 10 11, especially from about 10 6to about 10 10, more particularly from about 10 6to about 10 9, more particularly from about 10 6to about 10 8, the most especially from about 10 6to about 10 7colony-forming units (CFU).
15. salmonella attenuation mutants as described in any one of claim 8 to 14, it can be used for individualized cancer immunization therapy, and described treatment comprises the tumor antigen expression pattern of assess patient and/or the step of interstitial antigen presentation pattern.
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